(a) Field of the Invention
This disclosure relates to technologies for direct measurement of activity of a bioactive substance in cell using nanowires. In particular, a method of measuring intracellular activity of a bioactive substance using a support having nanowires to which a cell is immobilized and another support having nanowires to which a target substance for a bioactive substance to be detected is immobilized, and a chip for measuring intracellular activity of a bioactive substance including a support having nanowires to which a cell is immobilized and another support having nanowires to which a targeting substance for a bioactive substance to be detected are immobilized, are provided.
(b) Description of the Related Art
In general, enzyme activity has been studied using purified enzymes and substrates, and the methods include spectrophotometric assays measuring absorbance changes according to changes of substrates due to enzyme activities to identify enzyme activities, fluorometric assays measuring enzyme activities using differences in fluorescence between a product produced by enzyme activity and an initial substrate, calorimetry assays measuring heat absorbed or emitted by chemical reactions to measure changes of substrates due to enzyme reactions using microcalorimeter, chemiluminescent assays measuring light emission due to chemical reactions to measure enzyme activities, chromatographic assays measuring a product generated by enzyme activity using chromatography, and radiometric assays identifying enzyme activities using radioisotopes as substrates, and the like.
Since most of the methods for measuring enzyme activity are conducted in vitro using purified enzymes and substrates, various external conditions (ionic strength, salt concentration, temperature, pH, enzyme concentration, and etc.) may be controlled to conduct assays under best conditions for enzyme activity, which differs from endogenous enzyme conditions in cells. Since the existence of large quantities of macromolecules in cells may influence functional and structural stability of protein, and enzyme reaction including rate of reaction, the result of activity analysis using purified enzyme cannot always be regarded as representing enzyme action in cells.
Moreover, since in many cases, another enzyme reversing the reaction of one enzyme exists (for example, protein kinase and phosphatase which respectively cause phosphorylation and dephosphorylation exist simultaneously in cells), enzyme activity in the cell context may not be predicted simply by in vitro cell activity analysis. Recently, for measurement of endogenous enzyme activity in cells, various methods involving instantaneous cell lysis followed by assays have been extensively employed. However, the possibility that enzyme environment and activity may be changed during the cell lysis and lysate preparation cannot be excluded, and thus a method for analysis of enzyme activity with maintaining cells alive is required.
Nanotechnology enables characterization and control of material in atomic and molecular unit, and it has recently been combined with biotechnology and developed as new technology for future. Many study results have been reported on low dimensional nanostructures, and particularly one-dimensional nanomaterials such as nanorods, nanowires, nanotubes, nanobelts and the like with excellent optical, electrical, chemical and physical properties have been applied in various fields.
So far, biomedical applications of nanotechnology have mainly been focused on elucidating characteristics of nanoparticles for bio-imaging technology, biosensor, and local drug delivery. Since early 2000's, an intracellular drug delivery technique named nanotube spearing using nanomaterial supernatants has been developed. In 2007, Peidong Yang et al. of UC Berkeley, U.S.A. reported a study result of incubation of mice embryonic stem cells using vertically grown silicon nanowires and DNA transfection upon penetrating cells with the nanowires (Kim W, Ng J K, Kunitake M E, Conklin B R, Yang P, J. (2007) Am. Chem. Soc. 128:8990-8991), and suggested a possibility of application of vertically grown nanowires to live cells for the first time, although the transfection efficiency was very low as less than 1%.